Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

• Published in: Viruses 2021-08, Vol.13 (9), p.1723
• Main Authors: Chey, Soroth, Palmer, Juliane Maria, Doerr, Laura, Liebert, Uwe Gerd
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophage T7 - genetics; Chlorocebus aethiops; Cloning; Complementary DNA; Cytoplasm; DNA viruses; DNA, Complementary; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - metabolism; Efficiency; Enzymes; Gene transfer; Genetics; Genome, Viral; Genomes; Hepatitis; Humans; Measles; measles virus; Measles virus - genetics; multivalent vaccines; negative-strand RNA virus; Nucleotides; oncolytic vectors; Phages; Plasmids; Promoter Regions, Genetic; Promoters; Proteins; Rabies; Reassortant Viruses - genetics; recombinant virus; reverse genetics; Reverse Genetics - methods; Ribozymes; RNA polymerase; RNA viruses; RNA, Viral - genetics; Transcription; Vaccines; Vero Cells; Viral Proteins - metabolism; Viruses
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v13091723

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.